Processes for calculating phased fetal genomic sequences

ABSTRACT

The present invention provides processes for calculating phased genomic sequences of the fetal genome using fetal DNA obtained from a maternal sample. The processes and systems of the present invention utilize novel technological and computational approaches to detect fetal genomic sequences and determine the phased heritable genomic sequences. The invention could be used, e.g., to identify in utero deleterious mutations carried by the parents and inherited by a fetus within a particular heritable genomic region.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of provisional Patent Application Ser. No. 61/649,445, filed May 21, 2012 and is incorporated herein by reference.

FIELD OF THE INVENTION

The invention provides processes for calculating phased genomic information for a fetus using maternal samples including maternal blood, plasma and serum.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and processes will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and processes referenced herein do not constitute prior art under the applicable statutory provisions.

An individual's genetic profile plays an important role in determining risk for disease and response to medical therapy. The development of technologies that facilitate rapid whole-genome sequencing will provide unprecedented power in the estimation of disease risk. Improvements in sequencing technology have enabled cost effective generation of whole genome sequences for individuals. By combining whole genome sequence information with family or pedigree information or with longer sequencing read technology, one may also now phase genomes. A phased genome will describe which variants are aggregated together within chromosomal regions for a particular individual. The interrogation of the entire phased genome provides superior sensitivity to linked genetic features and identification of recombination events.

It has been long recognized that certain sources of biological samples from a pregnant mammal (e.g., blood or plasma), contains DNA from both the mother and the fetus. This recognition has led to the use of maternal samples to identify, non-invasively to the fetus, fetal genetic characteristics, including qualitative (e.g., sex determination and RhD status) and quantitative (fetal copy number variations including trisomies) genetic detection of fetal sequences (for review see, e.g., Lo et al., October 2011). It has also been demonstrated by deep sequencing of the cell-free DNA in a maternal sample that sequences representative of the entire fetal genome is present in circulation. (Lo Y-M et al., Sci Transl Med. 2010 Dec. 8; 2(61):61ra91.) However, the percent fetal DNA is usually present in a low amount, usually 3-40%. Although deep, whole-genome sequencing of the fetal genome has been performed, with conventional technologies this approach is at present economically infeasible for widespread clinical or commercial use.

Thus, improved processes and systems for the identification of inherited alleles in a fetus from a maternal sample would be of great benefit in the art. The present invention addresses this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.

The present invention provides processes for calculating phased genomic sequences of the fetal genome using fetal DNA obtained from a maternal sample. The processes and systems of the present invention utilize novel technological and computational approaches to detect fetal genomic sequences and determine the phased heritable genomic sequences. The invention could be used, e.g., to identify in utero deleterious mutations carried by the parents and inherited by a fetus within a particular heritable genomic region.

The processes of the invention provide methods for “phasing” the fetal DNA, i.e. determining the nucleic acids that are heritably linked within a single genomic region, e.g., a chromosome. The phased data from the fetal sequences can be used to determine whether the fetus is at risk for many diseases, disorders and/or predispositions based on the inheritance of one or more heritable genomic regions present in the fetal genome.

In one aspect, the processes and systems of the invention utilize chromosome-specific genomic sequence information from the mother and/or father, and preferably both the mother and father. In a preferred embodiment, the processes and systems utilize phased, whole-genome, chromosome-specific information of both the mother and father. This sequence information from the mother and father may be obtained through whole genome sequencing or via other methods, e.g., array hybridization followed by phasing of the sequence data from the mother and/or father. Such knowledge of the parental genomes can be used to determine which clinically or phenotypically significant alleles are inherited in the fetus within a heritable genomic region.

In one aspect, the invention provides a computer-implemented process for determining the phased composition in a fetal heritable genomic region, comprising the steps of: providing phased sequence information from at least one corresponding parental heritable genomic region; identifying five or more informative loci in the fetal DNA from a maternal sample corresponding to a heritable genomic region of interest; determining the paternal contribution of the heritable genomic region of interest based on the identified paternal contribution of the five or more informative loci; calculating the maternal contribution of the heritable genomic region based on the determined paternal contribution and the five or more informative loci; and predicting the likely phased composition of the phased fetal heritable genomic region based on the maternal and paternal contributions of the heritable genomic region.

In another aspect, the invention provides a computer-implemented process for determining the phased composition in a fetal heritable genomic region, comprising the steps of: providing phased sequence information from the maternal and paternal genome on at least one corresponding heritable genomic region; masking sequence information on loci that are indistinguishable between the maternal and paternal genome; providing empirical sequence information on five or more informative loci from a maternal sample corresponding to the heritable genomic region; calculating the predicted paternal contribution of the heritable genomic region to the fetus based on the empirically identified paternal sequences of the five or more informative loci; calculating the predicted maternal contribution of the heritable genomic region to the fetus based on the ratio of empirically identified sequences in the maternal sample; and providing a likelihood value of the fetal heritable genomic region contributed by the maternal and/or paternal source based on the predicted maternal and paternal contributions of the heritable genomic region to the fetus.

In yet another aspect, the invention provides a computer-implemented process to calculate a value of likelihood for a fetal heritable genomic region, comprising: providing maternal and paternal sequence information for a heritable genomic region; providing empirical sequence information from a heritable genomic region within a maternal sample, wherein the sequence information is obtained from the maternal sample using massively parallel sequencing; identifying at least five informative loci within the maternal and paternal heritable genomic region; calculating a value of the likelihood of the heritable genomic region inherited by the fetus from the father based on the informative loci and the empirical sequence information; identifying at least five loci which are maternally and paternally heterozygous; and calculating a value of the likelihood of the heritable genomic region inherited by the fetus from the mother based on value of the likelihood of the heritable genomic region inherited by the fetus from the father and the empirical sequence information on the loci which are maternally and paternally heterozygous.

In a preferred aspect of the invention, the fetal genetic variation within one or more heritable genomic regions can be imputed from a subset of parental informative loci within the heritable genomic regions. Thus, identifying certain alleles in the fetal genome can allow information of alleles that are not directly detected to be imputed from those that are detected. In this way, the heritable genomic regions in the fetus can be identified from a subset of informative loci, and preferably five or more informative loci, within the paternally-inherited and maternally-inherited heritable genomic regions.

In some aspects the maternal sample is a cell free maternal sample, and preferably maternal plasma or serum. In other aspects, the maternal sample comprises fetal cells.

In preferred aspects, the phased sequence information of parental genome is provided by sequencing, and more preferably whole genome sequencing. Preferably this is accomplished using long-read sequencing technologies that are more effective in providing phased information, or by combining short-read with long-read sequencing technologies. When combining sequencing technologies, the short-read coverage of the genome is preferably 20× or greater and the long-read sequencing coverage of the genome is preferably less than 5×. In other aspects, the phased allelic sequence information of the corresponding parental heritable genomic region is determined in part by pedigree analysis.

Generally, the allelic sequence information from the fetus comprises sequence information from at least twenty informative loci in the heritable genomic region, although as few as five informative loci can be used. In some aspects, the allelic sequence information from the fetus comprises sequence information on at least fifty informative loci in the heritable genomic region. In more specific aspects, the allelic sequence information from the fetus comprises sequence information on at least one hundred informative loci in the heritable genomic region.

In certain aspects, phasing of the fetal nucleic acids is performed for a sub-chromosomal unit. In other aspects, phasing of the fetal nucleic acids is performed for an entire chromosome. In yet other aspects, phasing of the fetal nucleic acids is performed for multiple fetal chromosomes. In still other preferred aspects, it is performed for the entire fetal genome.

In some aspects, the fetal genomes are analyzed using sequence determination of fetal sequences, and assembly of heritable regions is performed via comparison to one or more external reference sequences. In some aspects, significant variants are grouped by chromosome and haplotype association to determine which groups of variants are associated in a heritable region.

It is a feature of the invention that the source of the fetal DNA can be cell-free DNA obtained from maternal plasma or serum, and the processes of the invention identifies the fetal phasing in the background of the maternal DNA. The background maternal DNA contribution in the maternal sample can be “removed” from consideration either biochemically, through sensitive detection and comparison of the frequency of haplotypes present in the cell-free DNA, and/or via analytical analysis.

In some aspects of the invention, the processes utilize information on the fetal contribution of both the maternal genome and the paternal genome in the calculation of the phased fetal genomic regions.

In a preferred aspect, both maternal and paternal genomic information is used in the analysis of the fetal genome.

In other aspects, the association of significant variants in a fetal heritable region is determined by sequencing, and preferably massively parallel sequencing followed by allelic assembly.

DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram illustrating the difference between identification of fetal alleles and phased allelic information.

FIG. 2 is a chart illustrating informative loci.

FIG. 3 is first illustration of fetal phased allelic chromosomes based on the maternal and paternal genotyping.

FIG. 4 is a second illustration of fetal phased allelic chromosomes based on the maternal and paternal genotyping.

FIG. 5 is a third illustration of fetal phased allelic chromosomes based on the maternal and paternal genotyping.

FIG. 6 is a block diagram illustrating an exemplary system environment.

DETAILED DESCRIPTION OF THE INVENTION

The processes described herein may employ, unless otherwise indicated, conventional techniques and descriptions of molecular biology (including recombinant techniques), genomics, biochemistry, and sequencing technology, which are within the skill of those who practice in the art. Such conventional techniques include hybridization and ligation of oligonucleotides, next generation sequencing, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the examples herein. However, equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Green, et al., Eds., Genome Analysis: A Laboratory Manual Series (Vols. I-IV) (1999); Weiner, et al., Eds., Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler, Eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNA Microarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics: Sequence and Genome Analysis (2004); Sambrook and Russell, Condensed Protocols from Molecular Cloning: A Laboratory Manual (2006); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual (2002) (all from Cold Spring Harbor Laboratory Press); Stryer, L., Biochemistry (4th Ed.) W.H. Freeman, New York (1995); Gait, “Oligonucleotide Synthesis: A Practical Approach” IRL Press, London (1984); Nelson and Cox, Lehninger, Principles of Biochemistry, 3^(rd) Ed., W.H. Freeman Pub., New York (2000); and Berg et al., Biochemistry, 5^(th) Ed., W.H. Freeman Pub., New York (2002), all of which are herein incorporated by reference in their entirety for all purposes. Before the present compositions, research tools and processes are described, it is to be understood that this invention is not limited to the specific processes, compositions, targets and uses described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to limit the scope of the present invention, which will be limited only by appended claims.

It should be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a nucleic acid region” refers to one, more than one, or mixtures of such regions, and reference to “an assay” includes reference to equivalent steps and processes known to those skilled in the art, and so forth.

Where a range of values is provided, it is to be understood that each intervening value between the upper and lower limit of that range—and any other stated or intervening value in that stated range—is encompassed within the invention. Where the stated range includes upper and lower limits, ranges excluding either of those included limits are also included in the invention.

Unless expressly stated, the terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art. The following definitions are intended to aid the reader in understanding the present invention, but are not intended to vary or otherwise limit the meaning of such terms unless specifically indicated. All publications mentioned herein, and in particular patent applications and issued patents, are incorporated by reference for the purpose of describing and disclosing various aspects, details and uses of the processes and systems that are described in the publication and which might be used in connection with the presently described invention.

In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

DEFINITIONS

The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art. The following definitions are intended to aid the reader in understanding the present invention, but are not intended to vary or otherwise limit the meaning of such terms unless specifically indicated.

The term “amplified nucleic acid” is any nucleic acid molecule whose amount has been increased at least two fold by any nucleic acid amplification or replication process performed in vitro as compared to the starting amount in a maternal sample.

The term “diagnostic tool” as used herein refers to any composition or assay of the invention used in combination as, for example, in a system in order to carry out a diagnostic test or assay on a patient sample.

The term “DNA contribution” refers to the percentage, proportion or measurement such as weight by volume of nucleic acid in a sample that is contributed by a source, such as the mother or a fetus.

The term “extrinsic factor” includes any information pertinent to the calculation of an odds ratio that is not empirically derived through detection of a maternal and fetal locus. Examples of such extrinsic factors include information related to maternal age, information related to gestational age, information related to previous pregnancies with an aneuploid fetus, previous serum screening results, ultrasound findings and the like. In preferred embodiments, the step of calculating and/or adjusting the computed odds ratio uses extrinsic factors related to both maternal age and gestational age.

The term “genetic feature” includes any feature within a genome that is identifiable using, e.g., techniques such as sequence determination or hybridization. Genetic features include, but are not limited to, single nucleotide polymorphisms, tandem single nucleotide polymorphisms, short tandem repeats, expansions (e.g., triplet code repeats), methylation patterns, and the like.

The term “heritable region” as used herein includes any larger portion of DNA from a single allele that can be elucidated using conventional phasing technologies available to those in the art. In certain preferred aspects, the heritable region is a chromosome. In most preferred aspects, multiple heritable regions are detected, and most preferably thus includes a subset of the chromosomes from a parent, and in a more preferred aspect all of the chromosomes inherited from a parent.

The term “hybridization” generally means the reaction by which the pairing of complementary strands of nucleic acid occurs. DNA is usually double-stranded, and when the strands are separated they will re-hybridize under the appropriate conditions. Hybrids can form between DNA-DNA, DNA-RNA or RNA-RNA. They can form between a short strand and a long strand containing a region complementary to the short one. Imperfect hybrids can also form, but the more imperfect they are, the less stable they will be (and the less likely to form).

The terms “locus” and “loci” as used herein refer to a nucleic acid region of known location in a genome.

The term “informative locus” as used herein refers to a locus or pair of loci with one or more distinguishing regions useful in determining the phasing of a fetal heritable region.

The term “maternal sample” as used herein refers to any sample taken from a pregnant mammal which comprises a maternal source and a fetal source of nucleic acids (e.g., RNA or DNA).

The term “non-maternal” allele means an allele with a polymorphism and/or mutation that is found in a fetal allele (e.g., an allele with a de novo SNP or mutation) and/or a paternal allele, but which is not found in the maternal allele.

The term “phasing” as used herein refers to determination of genetic features which are located within a heritable region, e.g., the alleles that reside in a particular genomic region of a chromosome. For example, phasing can be performed on an entire chromosome to determine which genetic features will be heritably linked. Phasing thus provides the ability to distinguish which alleles belong to which chromosome, and to identify which alleles will be inherited together upon meiosis.

As used herein “polymerase chain reaction” or “PCR” refers to a technique for replicating a specific piece of target DNA in vitro, even in the presence of excess non-specific DNA. Primers are added to the target DNA, where the primers initiate the copying of the target DNA using nucleotides and, typically, Taq polymerase or the like. By cycling the temperature, the target DNA is repetitively denatured and copied. A single copy of the target DNA, even if mixed in with other, random DNA, can be amplified to obtain billions of replicates. The polymerase chain reaction can be used to detect and measure very small amounts of DNA and to create customized pieces of DNA. In some instances, linear amplification processes may be used as an alternative to PCR.

The term “polymorphism” as used herein refers to any genetic characteristic in a locus that may be indicative of that particular locus, including but not limited to single nucleotide polymorphisms (SNPs), methylation differences, short tandem repeats (STRs), and the like.

The term “polymorphic locus” as used herein refers to a locus with two or more detectable alleles within a population. Generally, a polymorphic locus will have the most common allele less than 70%.

Generally, a “primer” is an oligonucleotide used to, e.g., prime DNA extension, ligation and/or synthesis, such as in the synthesis step of the polymerase chain reaction or in the primer extension techniques used in certain sequencing reactions. A primer may also be used in hybridization techniques as a means to provide complementarity of a nucleic acid region to a capture oligonucleotide for detection of a specific nucleic acid region.

The term “research tool” as used herein refers to any composition or assay of the invention used for scientific enquiry, academic or commercial in nature, including the development of pharmaceutical and/or biological therapeutics. The research tools of the invention are not intended to be therapeutic, to be diagnostic or to be subject to regulatory approval; rather, the research tools of the invention are intended to facilitate research and aid in such development activities, including any activities performed with the intention to produce information to support a regulatory submission.

The term “selected nucleic acid region” as used herein refers to a nucleic acid region corresponding to a genomic region on an individual chromosome. Such selected nucleic acid regions may be directly isolated and enriched from the sample for detection, e.g., based on hybridization and/or other sequence-based techniques, or they may be amplified using the sample as a template prior to detection of the sequence. Nucleic acids regions for use in the processing systems of the present invention may be selected on the basis of DNA level variation between individuals, based upon specificity for a particular chromosome, based on CG content and/or required amplification conditions of the selected nucleic acid regions, or other characteristics that will be apparent to one skilled in the art upon reading the present disclosure.

The terms “sequencing”, “sequence determination” and the like as used herein refers generally to any and all biochemical processes that may be used to determine the order of nucleotide bases in a nucleic acid.

The term “specifically binds”, “specific binding” and the like as used herein, refers to one or more molecules (e.g., a nucleic acid probe or primer, antibody, etc.) that bind to another molecule, resulting in the generation of a statistically significant positive signal under designated assay conditions. Typically the interaction will subsequently result in a detectable signal that is at least twice the standard deviation of any signal generated as a result of undesired interactions (background).

The term “value of the likelihood” refers to any value achieved by directly calculating likelihood or any value that can be correlated to or otherwise indicative of a likelihood.

The term “value of the probability” refers to any value achieved by directly calculating probability or any value that can be correlated to or otherwise indicative of a probability.

THE INVENTION IN GENERAL

The present invention provides methods for identifying the particular alleles in a fetal genome using a subset of allelic information from the fetus using a maternal sample and a determination of the phased genomic data of the mother and/or father. Phased data provides information not just on the genotype of the parent (i.e., the two alleles that are inherited for a particular genomic region), but also the organization of the genetic information (e.g., the haplotypes that are linked on a particular chromosome).

As a parent generally passes one of the two copies of each chromosome on to their offspring, the genes received by a child are typically heritably linked since they are located on the same chromosome. Knowledge of the phased genomic information of the parents allows a subset of alleles to be samples in the fetal genome to identify the likelihood that a fetus has inherited a particular chromosome from the mother and/or father.

The fetal genotypes are determined from a maternal sample, preferably cell-free DNA from a maternal blood sample. In one example, one determines the fetal genotype where the mother is homozygous and the fetus is heterozygous. In those instances, one identifies the “minor” allele. In another example, one determines the fetal genotype where the mother is heterozygous and the fetus is homozygous. This may be done by first genotyping the mother from a pure cellular sample and then comparing that genotype to that of the genotype from the maternal sample to observe a shift in the expected counts.

In one example, the maternal sample is genotyped at more than 5,000 locations in all chromosomes. In another example the sample is genotyped at more than 10,000 locations in all chromosomes. In another example, the sample is genotyped at more than 20,000 locations in all chromosomes. In another example, the sample is genotyped at more than 50,000 locations in all chromosomes. In another example, the sample is genotyped at more than 100,000 locations in all chromosomes.

The genotyping may be done with many different assays and detection platforms. With respect to preferred genotyping assays, one that facilitates high multiplexing is desirable.

In a preferred aspect, the maternal and fetal DNA is interrogated using sequence determination of universally amplified sequences. In certain aspects, this utilizes one of the following combined selective and universal amplification techniques: (1) LDR coupled to PCR; (2) primary PCR coupled to secondary PCR coupled to LDR; and (3) primary PCR coupled to secondary PCR. Each of these aspects of the invention has particular applicability in detecting certain nucleic acid characteristics. However, each requires the use of coupled reactions for multiplex detection of nucleic acid sequence differences where oligonucleotides from an early phase of each process contain sequences which may be used by oligonucleotides from a later phase of the process.

Barany et al., U.S. Pat. Nos. 6,852,487, 6,797,470, 6,576,453, 6,534,293, 6,506,594, 6,312,892, 6,268,148, 6,054,564, 6,027,889, 5,830,711, 5,494,810, describe the use of the ligase chain reaction (LCR) assay for the detection of specific sequences of nucleotides in a variety of nucleic acid samples.

Barany et al., U.S. Pat. Nos. 7,807,431, 7,455,965, 7,429,453, 7,364,858, 7,358,048, 7,332,285, 7,320,865, 7,312,039, 7,244,831, 7,198,894, 7,166,434, 7,097,980, 7,083,917, 7,014,994, 6,949,370, 6,852,487, 6,797,470, 6,576,453, 6,534,293, 6,506,594, 6,312,892, and 6,268,148 describe the use of the ligase detection reaction with detection reaction (“LDR”) coupled with polymerase chain reaction (“PCR”) for nucleic acid detection.

Barany et al., U.S. Pat. Nos. 7,556,924 and 6,858,412, describe the use of padlock probes (also called “precircle probes” or “multi-inversion probes”) with coupled ligase detection reaction (“LDR”) and polymerase chain reaction (“PCR”) for nucleic acid detection.

Barany et al., U.S. Pat. Nos. 7,807,431, 7,709,201, and 7,198, 814 describe the use of combined endonuclease cleavage and ligation reactions for the detection of nucleic acid sequences.

Willis et al., U.S. Pat. Nos. 7,700,323 and 6,858,412, describe the use of precircle probes in multiplexed nucleic acid amplification, detection and genotyping

Ronaghi et al., U.S. Pat. No. 7,622,281 describes amplification techniques for labeling and amplifying a nucleic acid using an adapter comprising a unique primer and a barcode.

In addition to the various amplification techniques, numerous methods of sequence determination are compatible with the processes and systems of the inventions. Preferably, such methods include “next generation” methods of sequencing. Exemplary methods for sequence determination include, but are not limited to, hybridization-based methods, such as disclosed in Drmanac, U.S. Pat. Nos. 6,864,052; 6,309,824; and 6,401,267; and Drmanac et al, U.S. patent publication 2005/0191656, which are incorporated by reference, sequencing by synthesis methods, e.g., Nyren et al, U.S. Pat. Nos. 7,648,824, 7,459,311 and 6,210,891; Balasubramanian, U.S. Pat. Nos. 7,232,656 and 6,833,246; Quake, U.S. Pat. No. 6,911,345; Li et al, Proc. Natl. Acad. Sci., 100: 414-419 (2003); pyrophosphate sequencing as described in Ronaghi et al., U.S. Pat. Nos. 7,648,824, 7,459,311, 6,828,100, and 6,210,891;, and ligation-based sequencing determination methods, e.g., Drmanac et al., U.S. Pat. Appln No. 20100105052, and Church et al, U.S. Pat. Appln Nos. 20070207482 and 20090018024.

Alternatively, nucleic acid regions of interest can be selected and/or identified using hybridization techniques. Methods for conducting polynucleotide hybridization assays for detection of have been well developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Maniatis et al. Molecular Cloning: A Laboratory Manual (2^(nd) Ed. Cold Spring Harbor, N.Y., 1989); Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, Calif., 1987); Young and Davis, P.N.A.S, 80: 1194 (1983). Methods and apparatus for carrying out repeated and controlled hybridization reactions have been described in U.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which are incorporated herein by reference

The present invention also contemplates signal detection of hybridization between ligands in certain preferred aspects. See U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and 6,225,625, in U.S. Patent application 60/364,731 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensity data are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839, 5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723, 5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030, 6,201,639; 6,218,803; and 6,225,625, in U.S. Patent application 60/364,731 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.

Multiplexed PCR and array-based pull-outs are alternative options. With respect to detection platforms, the most preferred option is high-throughput DNA sequencing such as with Illumina, Complete Genomics and Ion Torrent. Array and qPCR read-outs are also possibilities. After the fetal genotypes have been determined, one compares those fetal genotypes to the phased parental genotypes. By using the haplotype information, one can identify alleles that would have been inherited together, thus identifying which chromosome or portions of chromosomes have been inherited. Once one has identified which chromosome or portions of chromosome have been inherited, one can then impute the fetal sequence. In the case where portions of chromosomes have been inherited, the sequence information between those portions is indeterminate. The amount of indeterminate sequence information is highly dependent upon the number of genotypes determined. Increasing the number of genotypes decreases the amount of indeterminate sequence information as one can determine with more certainty where the recombination site occurred. After the imputation of the fetal sequence, one may determine which clinically or phenotypically significant variants the fetus has inherited from each parent. It is important to note that one does not actually have to determine the fetal variant of clinical significance directly in the maternal sample. This can be done by imputing the variant from knowing the inheritance of other variants.

The processes and systems of the present invention utilize sequence information from heritable regions of the maternal and paternal genome to “phase” the fetal DNA obtained from a maternal source to obtain haplotype information for the heritable region of DNA. The parental genotypes for the heritable regions are determined using sequencing, and the linked alleles are identified through the sequencing process. As the fetal DNA may be available in the maternal sample as shorter regions (e.g., cell free DNA fragments), phasing of the fetal DNA may be more cost-effective than deep sequencing and assembly of the fetal genome.

Sequence information may be determined using methods that determine many (typically thousands to billions) of nucleic acid sequences in an intrinsically parallel manner, where many sequences are read out preferably in parallel using a high throughput serial process. Such methods include but are not limited to pyrosequencing (for example, as commercialized by 454 Life Sciences, Inc., Branford, Conn.); sequencing by ligation (for example, as commercialized in the SOLiD™ technology, Life Technology, Inc., Carlsbad, Calif.); sequencing by synthesis using modified nucleotides (such as commercialized in TruSeq™ and HiSeq™ technology by Illumina, Inc., San Diego, Calif., HeliScope™ by Helicos Biosciences Corporation, Cambridge, Mass., and PacBio RS by Pacific Biosciences of California, Inc., Menlo Park, Calif.), sequencing by ion detection technologies (Ion Torrent, Inc., South San Francisco, Calif.); sequencing of DNA nanoballs (Complete Genomics, Inc., Mountain View, Calif.); nanopore-based sequencing technologies (for example, as developed by Oxford Nanopore Technologies, LTD, Oxford, UK), and like highly parallelized sequencing methods.

In some aspects, the fetal haplotypes are inferred by haplotype resolution or haplotype phasing techniques. These methods work by applying the observation that certain haplotypes are common in certain genomic regions. Therefore, given a set of possible haplotype resolutions, these methods choose those that use fewer different haplotypes overall.

In specific aspects, combinatorial approaches (e.g., parsimony) are used for haplotype phasing. In brief, haplotypes for an individual are selected among competing possible haplotpes and the one that offers the simplest explanation of the data derived from the fetal DNA is used to identify the most likely haplotypes for a heritable region.

In other specific aspects, likelihood functions are used. For example, haplotype phasing can be determined based on models and assumptions such as those that utilize genetic equilibrium (e.g., the Hardy-Weinberg principle). In the simplest case of a single locus with two alleles: the dominant allele is denoted A and the recessive a and their frequencies are denoted by p and q; freq(A)=p; freq(a)=q; p+q=1. If the population is in equilibrium, then we will have freq(AA)=p² for the AA homozygotes in the population, freq(aa)=q² for the aa homozygotes, and freq(Aa)=2pq for the heterozygotes.

Other aspects employ retrospective models of population genetics, e.g., the coalescent theory model. Each of these models can be combined with optimization algorithms such as expectation-maximization algorithm (EM), Markov chain Monte Carlo (MCMC), or hidden Markov models (HMM).

An example is in the case of cystic fibrosis (CF) testing. By sequencing, one would know that perhaps one or more of the parents are a CF carrier. The parents would like to know whether the fetus has inherited one allele, in which case it may be a CF carrier, or whether the fetus has inherited two alleles, in which case it may be symptomatic for CF. By genotyping informative loci close to the CF gene in the maternal sample, one may determine which chromosomes were inherited by the fetus and thus which CF alleles the fetus inherited from each parent, determining the CF status of the fetus.

Current approaches for full-scale genomic phasing require too much sequencing to be cost-effective. The processes of the invention using phasing based on fetal DNA fragments would greatly reduce the amount of sequencing necessary to determine the fetal genome in utero.

Techniques for Phasing the Fetal Genome

There are many ways to phase a mammalian genome, including long-range sequencing (>1000 bp) to identify overlapping haplotype information, sequencing or genotyping of predecessors or descendants to determine which alleles were inherited together, and imputation by population-based haplotype information.

For the present invention, information from one or both parents makes it possible to phase the fetal genome (for the vast majority of SNP calls) using a maternal sample. The processes of the invention rely on the fact that for most situations, the alleles inherited from the maternal and/or paternal genome can be provided, and these can be used not only to identify the value of likelihood of a specific chromosome being inherited by the fetus, but also identification of recombination events and the genomic.

For example, as illustrated in FIG. 1, if at a particular position, a fetal genotype call is AB (101), the paternal genotype is AA, and the maternal genotype is BB, the fetal A allele must have come from the father, and the fetal B allele must have come from the mother. Such alleles where fetal phase can be determined are considered informative. The cumulative data can be processed to determine a value of likelihood of a particular chromosome being inherited by the fetus. The A allele from the father is associated with a certain paternally-inherited fetal chromosome 103 while the B allele is associated with a certain maternally-inherited fetal chromosome 105. FIG. 2 provides examples of informative alleles based on maternal, paternal and fetal genotype that may be used in the processes of the invention.

FIG. 3 illustrates the utility of informative loci in determining the allelic make-up, and therefore phasing, of a fetal chromosome. The ability to test a limited number of allelic variants to infer all of the other alleles inherited by the fetus is a central concept of the invention. Thus, by determining the inheritance of the allels shown in bold, the processes of the invention allow an imputation of the entire allelic makeup of the parentally-inherited chromosomes. Data on the alleles of a maternal phased chromosome 301 and a paternal phased chromosome 303 are provided, and the linked haplotype data from these chromosomes used to identify the phasing of the corresponding inherited fetal chromosomes 305. In this particular illustration, the fetal chromosomes each have a recombination event resulting in individual inherited chromosomes having alleles inherited from both paternal and maternal chromosomes.

The informative loci from the maternal and paternal genome allow both identification of the likely chromosomes inherited and the identification of the recombination event. The resulting data can be used to determine a value of likelihood that the inherited fetal chromosomes comprise specific linked alleles in view of the recombination events. Using the maternal and paternal phased genomic information, the likely inherited paternally-derived chromosome 307 and the maternally-derived chromosome 309 inherited by the fetus can be calculated using parental data.

FIG. 4 demonstrates the instance in which heterozygosity between maternal and paternal alleles can be informative. For example, if both parents have the genotype AB, and the fetus has the genotype BB, then both parents must have contributed the B genotype to the fetus.

In other implementations, such as that illustrated in FIG. 5, the phasing of the fetal DNA can be only partially determined based on the allelic data of a maternal phased chromosome 501 and a paternal phased chromosome 503. This data can be used to identify the phasing of the corresponding inherited fetal chromosomes 505. As in FIG. 3, the fetal chromosome has a recombination event in the DNA inherited from both the paternal and the maternal genome. The linked allelic information in FIG. 5, however, is ambiguous regarding the exact location of the recombination event due to heterozygosity at the maternal and paternal alleles at the site of recombination, so a value of likelihood for the fetal chromosomes can be determined on opposite sides of the recombination event but only as provided based on the available informative loci.

As the distinct position of the recombination event is unclear, a value of probability can be calculated given different markers and the value of likelihood that a recombination event may have occurred in a specific region of the chromosome. Using the maternal and paternal phased genomic information, the paternally-inherited fetal chromosome 507 and the maternally-inherited fetal chromosome 509 can be calculated but more information needs to be obtained in the recombination region to determine the allelic composition of the region.

In a preferred aspect, the processes of the invention utilizes paternal genomic information, maternal genomic information, and empirically-derived data from a maternal sample that comprises both maternal (the “major source”) and fetal (“the minor source”) DNA. The computational process provides a removal of all paternal and maternal genomic data which is the same across parental alleles, i.e., in which all homozygous loci that are the same between the mother and father are removed from the data set. Next, loci that are informative for the paternal allele in the fetus (i.e., “minor source informative”) are determined, and alleles that are specific to the paternal source are identified. This can be calculated using the empirically-derived data from the maternal sample, using counts determined from the nucleic acids present representative of each allele present in the maternal sample. One example of this is the use of a binomial equation such as: Bin(A+B, X), where A is the empirically determined level of a first allele, B is the empirically determined level of a second allele, and X is a factor of the fetal contribution to the maternal sample. For example if the fetal contribution is approximately 10%, then X=0.5.

For the equation to have sufficient power, X>β, where β is the minimum level of fetal contribution in a sample. As a general rule, β should be 2 or greater, although this will also depend upon the number of informative loci used and the amount of parental information available to be used in the processes of the invention.

From this information, the paternal contribution can be inferred for multiple minor source haplotypes that are associated with the fetal chromosome inherited from the father.

Once the paternal alleles are identified for these regions, the maternal allele can be imputed based on the empirically-determined ratios of the nucleic acids representing the different alleles present in the maternal sample. If, for instance, the mother and father are both heterozygous for an allele, the maternally-inherited allele is the same as the paternally-inherited allele, and the fetal contribution X=0.5, then the identified nucleic acids representative of the allele in the maternal sample would be approximately 55/100 counts for that allele. If, however, maternally-inherited allele is the same as the paternally-inherited allele, and the fetal contribution X=0.5, then the identified nucleic acids representative of the allele in the maternal sample would be approximately 50/100 counts for that allele.

Empirical Determination of Fetal Contribution in a Maternal Sample

Determining which genetic loci are contributed to the fetus from paternal sources may in certain aspects utilize information on the fetal contribution in a maternal sample. The estimation of fetal DNA proportion in a maternal sample, provides information used to calculate statistically significant differences in dosages for alleles of interest, and thus collectively for heritable genomic regions of interest.

In certain aspects, determination of fetal polymorphisms requires targeted SNP and/or mutation analysis to identify the presence of fetal DNA in a maternal sample. In one preferred aspect, the percent fetal cell free DNA in a maternal sample can be quantified using multiplexed SNP detection based on knowledge of the maternal and/or paternal genotype. The selected polymorphic nucleic acid regions from the maternal sample (e.g., plasma) are amplified. In a preferred aspect, the amplification is universal; and in a preferred embodiment, the selected polymorphic nucleic acid regions are amplified in one reaction in one vessel. Each allele of the selected polymorphic nucleic acid regions is determined and quantified. In a preferred aspect, high throughput sequencing is used for such determination and quantification.

Identification of informative loci is accomplished by observing a high frequency of one allele (>80%) and a low frequency (<20% and >0.15%) of the other allele for a particular selected nucleic acid region. The use of multiple loci is particularly advantageous as it reduces the amount of variation in the measurement of the abundance of the alleles between loci. All or a subset of the loci that meet this requirement can used to determine fetal contribution through statistical analysis. In one aspect, fetal contribution is determined by summing the low frequency alleles from two or more loci together, dividing by the sum of the low and high frequency alleles and multiplying by two.

In one aspect, data from selected nucleic acid regions may be excluded if the data from the region appears to be an outlier due to experimental error or from idiopathic genetic bias within a particular sample. In another aspect, selected data from certain nucleic acid regions may undergo statistical or mathematical adjustment such as normalization, standardization, clustering, or transformation prior to summation or averaging. In another aspect, data from selected nucleic acid regions may undergo both normalization and data experimental error exclusion prior to summation or averaging.

In a preferred aspect, data from 12 or more nucleic acid regions or loci are used for the analysis. In another preferred aspect, data from 24 or more nucleic acid regions or loci are used for the analysis. In another preferred aspect, data from 48 or more loci are used for the analysis. In another aspect, one or more indices are used to identify the sample, the locus, the allele or the identification of the nucleic acid. Such indices are as is described in co-pending applications U.S. Ser. Nos. 13/205,490 and 13/205,570 hereby incorporated herein by reference in their entirety.

In one preferred aspect, the percentage fetal contribution in a maternal sample is quantified using tandem SNP detection in the maternal and fetal alleles. Techniques for identifying tandem SNPs in DNA extracted from a maternal sample are disclosed in Mitchell et al, U.S. Pat. No. 7,799,531 and U.S. Ser. Nos. 12/581,070, 12/581,083, 12/689,924, and 12/850,588. These references describe the differentiation of fetal and maternal loci through detection of at least one tandem single nucleotide polymorphism (SNP) in a maternal sample that has a different haplotype between the fetal and maternal genome. Identification and quantification of these haplotypes can be performed directly on the maternal sample and used to determine the fetal proportion of nucleic acids in the maternal sample.

Determination of Fetal DNA Content in a Maternal Sample Using Epigenetic Allelic Ratios

Certain genes have been identified as having epigenetic differences between the fetus and the mother, and such genes are candidate loci for fetal DNA markers in a maternal sample. See, e.g., Chim, et al., PNAS USA, 102:14753-58 (2005). These loci, which are unmethylated in the fetus but are methylated in maternal blood cells, can be readily detected in maternal plasma. The comparison of methylated and unmethylated amplification products from a maternal sample can be used to quantify the percent fetal DNA contribution to the maternal sample by calculating the epigenetic allelic ratio for one or more of such sequences known to be differentially-methylated in fetal DNA as compared to maternal DNA.

To determine methylation status of nucleic acids in a maternal sample, the nucleic acids of the sample are subjected to bisulfite conversion. Conventional processes for such bisulphite conversion include, but are not limited to, use of commercially available kits such as the Methylamp™ DNA Modification Kit (Epigentek, Brooklyn, N.Y.). Allelic frequencies and ratios can be directly calculated and exported from the data to determine the percentage of fetal DNA in the maternal sample.

Human Reference Sequences

One of the challenges to interpretation of genome sequence data is the assembly and variant calling of sequence reads against the human reference genome. Although de novo assembly of genome sequences from raw sequence reads represents an alternative approach, computational limitations and the large amount of mapping information encoded in relatively invariant genomic regions make this an unattractive option presently. The National Center for Biotechnology Information (NCBI) human reference genome (Pruitt K D et al., Nucleic Acids Res. 2012 January; 40(Database issue):D130-5. Epub 2011 Nov. 24) is derived from DNA samples from a small number of anonymous donors and therefore represents a small sampling of the broad array of human genetic variation. For purposes of more diverse populations (or populations of specific descent) or more tailored genomes (individual genomes or cumulative reference of multiple genomes).

In some aspects of the invention, synthetic human reference sequences that are ethnically concordant with a pregnant subject and her family are used for the analysis of genomes from a nuclear family. Such reference sequences are described, e.g., in Dewey F E et al., PLoS Genet. 2011 September; 7(9):e1002280. Epub 2011 Sep. 15. The use of a major allele reference sequence results in improved genotype accuracy for variant loci. Recombination sites can be inferred to the lowest median resolution demonstrated to date (<1,000 base pairs).

Determination of the whole genome sequence of the mother and fetus, and preferably the mother, father and fetus allows multigenic risk for inherited diseases and disorders, and may also be useful in optimizing pharmaceutical intervention based on metabolism or predicted response to various drugs. These ethnicity-specific, family-based approaches to interpretation of genetic variation are emblematic of the next generation of genetic risk assessment using whole-genome sequencing.

Computer Implementation of the Processes of the Invention

FIG. 6 is a block diagram illustrating an exemplary system environment 60 in which the processes of the present invention may be implemented for calculating chromosome or loci dosage and fetal DNA contribution. The system 60 includes a server 62 and a computer 66. The computer 66 may be in communication with the server 62 through the same or different network 68.

According to the exemplary embodiment, the computer 66 executes a software component 64 that calculates fetal phased genomic information based on the provided data sets 74. In one embodiment, the computer 66 may comprise a personal computer, but the computer 66 may comprise any type of machine that includes at least one processor and memory.

The output of the software component 64 comprises a report 72 with a value of likelihood of inheritance of one or more heritable genomic regions. The report 72 may be paper that is printed out, or electronic, which may be displayed on a monitor and/or communicated electronically to users via e-mail, FTP, text messaging, posted on a server, and the like.

Although the process of the invention is shown as being implemented as software 64, it can also be implemented as a combination of hardware and software. In addition, the software 64 may be implemented as multiple components operating on the same or different computers.

Both the server 62 and the computer 66 may include hardware components of typical computing devices (not shown), including a processor, input devices (e.g., keyboard, pointing device, microphone for voice commands, buttons, touchscreen, etc.), and output devices (e.g., a display device, speakers, and the like). The server 62 and computer 66 may include computer-readable media, e.g., memory and storage devices (e.g., flash memory, hard drive, optical disk drive, magnetic disk drive, and the like) containing computer instructions that implement the functionality disclosed when executed by the processor. The server 62 and the computer 66 may further include wired or wireless network communication interfaces for communication.

While this invention is satisfied by aspects in many different forms, as described in detail in connection with preferred aspects of the invention, it is understood that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the specific aspects illustrated and described herein. Numerous variations may be made by persons skilled in the art without departure from the spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents. The abstract and the title are not to be construed as limiting the scope of the present invention, as their purpose is to enable the appropriate authorities, as well as the general public, to quickly determine the general nature of the invention. In the claims that follow, unless the term “means” is used, none of the features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. §112, ¶16. 

1. A computer-implemented process for determining the phased composition in a fetal heritable genomic region, comprising the steps of: providing phased sequence information from at least one corresponding parental heritable genomic region; identifying five or more informative loci in the fetal DNA from a maternal sample corresponding to a heritable genomic region of interest; determining the paternal contribution of the heritable genomic region of interest based on the five or more informative loci; calculating the maternal contribution of the heritable genomic region based on the determined paternal contribution and the five or more informative loci; and predicting the likely phased composition of the phased fetal heritable genomic region based on the maternal and paternal contributions of the heritable genomic region.
 2. The process of claim 1, wherein the maternal sample is a cell free maternal sample.
 3. The process of claim 2, wherein the cell free maternal sample is maternal plasma or serum.
 4. The process of claim 1, wherein the maternal sample comprises fetal cells.
 5. The process of claim 1, wherein the fetal genetic variation within the heritable genomic region is imputed from a subset of parental informative loci.
 6. The process of claim 1, wherein the phased sequence information of the heritable genomic region is determined by sequencing of the parental genome.
 7. The process of claim 1, wherein the phased sequence information of the corresponding parental heritable genomic region is determined by pedigree analysis.
 8. The process of claim 1, wherein the phased sequence information from the fetus comprises sequence information on at least twenty informative loci in the heritable genomic region.
 9. The process of claim 1, wherein the phased sequence information from the fetus comprises sequence information on at least fifty informative loci in the heritable genomic region.
 10. The process of claim 1, wherein the phased sequence information from the fetus comprises sequence information on at least one hundred informative loci in the heritable genomic region.
 11. The process of claim 1, wherein the heritable genomic region comprises a sub-chromosomal unit.
 12. The process of claim 1, wherein the heritable genomic region comprises an entire chromosome.
 13. The process of claim 1, wherein the heritable genomic region comprises the entire genome.
 14. A computer-implemented process for determining the phased composition in a fetal heritable genomic region, comprising the steps of: providing phased sequence information from the maternal and paternal genome on at least one corresponding heritable genomic region; masking sequence information on loci that are indistinguishable between the maternal and paternal genome; providing empirical sequence information on five or more informative loci from a maternal sample corresponding to the heritable genomic region; calculating the predicted paternal contribution of the heritable genomic region to the fetus based on the empirically identified paternal sequences of the five or more informative loci; calculating the predicted maternal contribution of the heritable genomic region to the fetus based on the ratio of empirically identified sequences in the maternal sample; and providing a likelihood value of the fetal heritable genomic region contributed by the maternal and/or paternal source based on the predicted maternal and paternal contributions of the heritable genomic region to the fetus.
 15. The process of claim 14, wherein the maternal sample is a cell free maternal sample.
 16. The process of claim 15, wherein the cell free maternal sample is maternal plasma or serum.
 17. The process of claim 14, wherein the maternal sample comprises fetal cells.
 18. The process of claim 14, wherein the phased sequence information of the heritable genomic region is determined by sequencing of the parental genome.
 19. The process of claim 14, wherein the fetal genetic variation within the heritable genomic region is imputed from a subset of parental informative loci.
 20. The process of claim 14, wherein the phased sequence information of the corresponding parental heritable genomic region is determined by pedigree analysis.
 21. The process of claim 14, wherein the phased sequence information from the fetus comprises sequence information on at least twenty informative loci in the heritable genomic region.
 22. The process of claim 14, wherein the phased sequence information from the fetus comprises sequence information on at least fifty informative loci in the heritable genomic region.
 23. The process of claim 14, wherein the phased sequence information from the fetus comprises sequence information on at least one hundred informative loci in the heritable genomic region.
 24. The process of claim 14, wherein the heritable genomic region comprises a sub-chromosomal unit.
 25. The process of claim 14, wherein the heritable genomic region comprises an entire chromosome.
 26. The process of claim 14, wherein the heritable genomic region comprises the entire genome.
 27. A computer-implemented process to calculate a value of likelihood for a fetal heritable genomic region, comprising: providing maternal and paternal sequence information for a heritable genomic region; providing empirical sequence information from a heritable genomic region within a maternal sample, wherein the sequence information is obtained from the maternal sample using massively parallel sequencing; identifying at least five informative loci within the maternal and paternal heritable genomic region; calculating a value of the likelihood of the heritable genomic region inherited by the fetus from the father based on the informative loci and the empirical sequence information; identifying at least five loci which are maternally and paternally heterozygous; calculating a value of the likelihood of the heritable genomic region inherited by the fetus from the mother based on value of the likelihood of the heritable genomic region inherited by the fetus from the father and the empirical sequence information on the loci which are maternally and paternally heterozygous.
 28. The process of claim 27, wherein the maternal sample is a cell free maternal sample.
 29. The process of claim 28, wherein the cell free maternal sample is maternal plasma or serum.
 30. The process of claim 27, wherein the maternal sample comprises fetal cells.
 31. The process of claim 27, wherein the phased sequence information of the heritable genomic region is determined by sequencing of the parental genome.
 32. The process of claim 27, wherein the fetal genetic variation within the heritable genomic region is imputed from a subset of parental informative loci.
 33. The process of claim 27, wherein the phased sequence information of the corresponding parental heritable genomic region is determined by pedigree analysis.
 34. The process of claim 27, wherein the phased sequence information from the fetus comprises sequence information on at least twenty informative loci in the heritable genomic region.
 35. The process of claim 27, wherein the phased sequence information from the fetus comprises sequence information on at least fifty informative loci in the heritable genomic region.
 36. The process of claim 27, wherein the phased sequence information from the fetus comprises sequence information on at least one hundred informative loci in the heritable genomic region.
 37. The process of claim 27, wherein the heritable genomic region comprises a sub-chromosomal unit.
 38. The process of claim 27, wherein the heritable genomic region comprises an entire chromosome.
 39. The process of claim 27, wherein the heritable genomic region comprises the entire genome. 